Part:BBa_K4927058
Protein scaffold
1. Mechanism of CE-Im protein reaction pairs
Colicin (colicin) is a non-specific nuclease, a group E bacterial toxin (E2, E7, E8 and E9), capable of non-specific degradation of nucleic acids to kill related microorganisms. CE nucleases consist of three functional domains, including receptor binding domain, membrane ectopic domain, and nuclease activity domain Taking CE7 as an example, Escherichia coli producing CE7 forms 1:1 non-covalent compounds with its homologous immune protein (Im7) to avoid suicide . Im7 can bind to external sites of immune proteins on CE7, and IM7-CE7 has a high affinity for protein interactions . Similarly, homologous orthogonal Im2-CE2 and Im9-CE9 also have strong protein-protein interaction, and their dissociation constants are in the range of 10-14~10-17 m . Although the three-dimensional spatial structure of Im8-CE8 complex has not been analyzed so far, we found that their structures are highly similar by comparing the sequences of CE2, CE7, CE8 and CE9. It can be inferred that the mechanism of action of Im2-CE2, Im7-CE7, Im8-CE8 and Im9-CE9 is similar.
2. construction of artificial protein scaffold system
The binding of the enzyme to the artificial protein scaffold depends on the tag fused by the two. The homologous and orthogonal CE-Im protein reaction pairs have high affinity and specific interaction, which is completely suitable for the construction of artificial protein scaffold system. However, CE nuclease is a kind of non-specific DNase, and its nuclease activity can affect the growth and development of its expressing cells. Therefore, when it is applied to artificial protein scaffold system, the nuclease activity of CE protein should be eliminated, while the specific binding ability between CE-IM should be preserved. In 2017, Vassylyeva et al. designed a truncated mutant CL7 with CE7 nuclease inactivation , and purified the high-efficiency CE7-IM7 protein reaction pair, which can be used for the assembly of artificial complexes in multi-enzyme cascade reaction systems.
1.Introduction of protein scaffold system
In living organisms, the survival and growth of each cell depends on the occurrence of thousands of different biological reactions. Biocatalysis can mediate intracellular transformation with high complexity, high efficiency and high selectivity. Enzymes, as catalysts for biological reactions, have evolved to be selective and specific enough to adapt to a variety of chemical transformations [1].
Much of the remarkable biocatalytic effect demonstrated by enzymes is due to a multi-step cascade process, and these unique characteristics depend on the three-dimensional spatial structure of the biocatalyst in a closed cellular environment to allow the formation of substrate channels in reaction intermediates [2]. However, in the case of low protein expression level and activity in the cell, enzymes that catalyze the reaction are assembled into multi-enzyme complexes to achieve multi-enzyme cascade [3], narrow the physical distance between the active sites of catalyst molecules to form substrate channels, enhance the synergistic effect between enzymes, and improve substrate utilization and product synthesis rate [4, 5].
In order to realize the multi-enzyme cascade metabolic pathway in vitro, more and more rational modified enzymes are used in the artificial cascade catalytic reaction to convert renewable resources into value-added products [6]. In the cascade catalytic reaction, the assembly of multi-enzyme complexes can be achieved through organometallic frameworks, fusion protein expression, self-assembly microstructures, and artificial biological scaffolds (Figure 1) [7-9]. Among them, the artificial biological scaffold system has obvious advantages over other systems, such as multiple types, high degrees of freedom, and high designability.
Figure 1. Multi-enzyme complex assembly for biocatalysis [2]. Note: Macromolecular scaffolds and engineered enzymes are used to simulate the intracellular biocatalysis cascade and the spatial structure of engineered enzymes in the multi-enzyme cascade system. E1, E2) : enzymes containing scaffolds; S1, P1) : substrates and products of E1; S2, P2) : substrates and products of E2.
Artificial biological scaffolds can be further divided into nucleic acid scaffolds, nucleic acid-protein scaffolds and protein scaffolds [10]. Nucleic acid scaffold (RNA/RNA, RNA/DNA and DNA/DNA double helix scaffold) is a supramolecular catalyst with control sequence and specified space assembled by hybridization of nucleic acid base sequence, which is not restricted by the number of protein binding sites, and can be artificially regulated by designing promoters and terminators. And bind to enzymes to form relatively complex multidimensional spatial structures [11]. However, the nature of nucleic acid scaffolds is very unstable, which is easy to be degraded by corresponding specific nuclease, and the multi-enzyme complex is easy to break the complex structure of DNA/RNA, which limits the application of nucleic acids in artificial scaffolds.
The artificial protein scaffold system constructed by using the communication between the orthogonal protein reaction pairs can co-locate multiple enzymes in a certain proportion and spatial order to form supramolecular complexes. The proximity of the active sites of multiple enzymes gives rise to the mechanism advantage, the rate-limiting reaction is accelerated, and the side reaction of the intermediate substrate channel is avoided, thus improving the catalytic efficiency of the enzyme and the utilization of the substrate [12]. At present, the typical protein scaffolders exist in the fibrils of microorganisms such as Clostridium cellulolyticum, which consist of the interacting Cohesin module and Dockerin module. By using this scaffold system, Liu Fang et al. constructed an artificial three-enzyme complex displayed on the surface of yeast, composed of methanol dehydrogenase (ADH), formaldehyde dehydrogenase (FALDH) and formate dehydrogenase (FDH), which can be used for methanol cascade catalytic complete oxidation to CO2. Compared with the free enzyme system, The NADH production rate of the three-enzyme cascade system was increased by 5 times [13, 14].
2.Mechanism of CE-Im protein reaction pairs
Colicin (colicin) is a non-specific nuclease, a group E bacterial toxin (E2, E7, E8 and E9), capable of non-specific degradation of nucleic acids to kill related microorganisms. CE nucleases consist of three functional domains, including receptor binding domain, membrane ectopic domain, and nuclease activity domain [15]. Taking CE7 as an example, Escherichia coli producing CE7 forms 1:1 non-covalent compounds with its homologous immune protein (Im7) to avoid suicide [16]. Im7 can bind to external sites of immune proteins on CE7, and IM7-CE7 has a high affinity for protein interactions [16, 17]. Similarly, homologous orthogonal Im2-CE2 and Im9-CE9 also have strong protein-protein interaction, and their dissociation constants are in the range of 10-14~10-17 m [18-21]. Although the three-dimensional spatial structure of Im8-CE8 complex has not been analyzed so far, we found that their structures are highly similar by comparing the sequences of CE2, CE7, CE8 and CE9. It can be inferred that the mechanism of action of Im2-CE2, Im7-CE7, Im8-CE8 and Im9-CE9 is similar.
3.construction of artificial protein scaffold system
The binding of the enzyme to the artificial protein scaffold depends on the tag fused by the two. The homologous and orthogonal CE-Im protein reaction pairs have high affinity and specific interaction, which is completely suitable for the construction of artificial protein scaffold system. However, CE nuclease is a kind of non-specific DNase, and its nuclease activity can affect the growth and development of its expressing cells. Therefore, when it is applied to artificial protein scaffold system, the nuclease activity of CE protein should be eliminated, while the specific binding ability between CE-IM should be preserved. In 2017, Vassylyeva et al. designed a truncated mutant CL7 with CE7 nuclease inactivation (FIG. 2), and purified the high-efficiency CE7-IM7 protein reaction pair, which can be used for the assembly of artificial complexes in multi-enzyme cascade reaction systems.
Figure 2. Construction of colicin E7 (CE7) truncated mutant (CL7). Note: a) : CE7/Im7 reaction pair; b) : CE7/DNA reaction pair; c) : protein sequence alignment between CE7 and CL7. Note: a): CE7 / Im7 reaction pair ; b): CE7 / DNA reaction pair ; c): CE7 and CL7 protein sequence alignment.
Based on the high similarity of the amino acid sequences of CE2, CE8, and CE9 to CE7, the truncated mutant CL7, which is inactivated by E. coli CE7 nuclease, was identified in our laboratory. The corresponding CE2, CE8, and CE9 truncated mutants CL2, CL8, and CL9 were designed, and the corresponding inhibitors Im2, Im7, Im8, and Im9 were obtained to construct the artificial protein scaffold Scaf-CIQ, as shown in Figure 3 [22]. This artificial protein scaffold has the advantages of excellent thermal stability, ultra-high affinity, high selectivity, small size and uniform structure of CL/Im protein interaction, etc., which can promote the formation of substrate channels, prevent the diffusion of intermediate products, and promote the timely transformation of intermediate products.
Figure 3. Schematic diagram of assembly of multi-enzyme complex synthesized by CL-Im quadruplex system [22]
4.Effect of artificial protein scaffold system on the activity of 4 enzymes
(1)The effect of artificial protein scaffold system on A2 activity
Methanol dehydrogenase CL2-ADH catalyzes the production of formaldehyde and NADH with methanol and NAD+ as substrates. In order to determine the activity of methanol dehydrogenase and the effect of scaffold on the activity of the enzyme, we used the methanol dehydrogenase liquid purified by Ni-NTA and ultrafiltered, and determined the protein concentration by Bradford method. As shown in Figure 4, in the whole reaction system, the final concentration of NAD+ was 0.1mM. We determined the concentration of NADH catalyzed by ADH-CL2 and ADH-CL2 + Scaf at different methanol concentrations. The results showed that the activity of ADH-CL2 was not affected after the addition of scaffold protein. It was worth noting that scaffold protein improved the catalytic efficiency of ADH-CL2 and the affinity between ADH-CL2 and the substrate.
Figure 4. Kinetic curve of methanol dehydrogenaseNote: a) : Kinetics curves of methanol dehydrogenase (CL2-ADH + Scaf) binding protein scaffolds; b) : kinetic curve of methanol dehydrogenase (CL2-ADH).
(2)Effect of artificial protein scaffold system on F7 activity
In the coenzyme regeneration system, formaldehyde dehydrogenase FALDH-CL7 is a rate-limiting enzyme, which determines the rate of the whole reaction. Therefore, optimizing the enzyme activity of FALDH-CL7 is essential for the sustained production of NADH. Using formaldehyde aqueous solutions of different concentrations as substrates, the generated NADH concentration was calculated by NADH standard curve, and the catalytic activities of FALDH-CL7 and FALDH-CL7+Scaf under different formaldehyde concentrations were measured and analyzed, as shown in FIG. 5.
Figure 5. Kinetic curve of formaldehyde dehydrogenase Note: a) : Kinetic curve of formaldehyde dehydrogenase (FALDH-CL7 + Scaf) binding protein scaffold; b) : kinetic curve of formaldehyde dehydrogenase (FALDH-CL7).
(3) The effect of artificial protein scaffold system on 8F activity
Formate dehydrogenase uses sodium formate and NAD+ as substrates to catalyze the production of CO2 and NADH. We determined the enzyme activity of formate dehydrogenase (CL8-FDH) and formate dehydrogenase (CL8-FDH+Scaf) bound to protein scaffold, and plotted the enzyme kinetics curve. Through the analysis and comparison of Km values of different enzymes, it was explored whether the addition of scaffold protein would affect the activity of formate dehydrogenase, and the results were shown in Figure 6.
Figure 6. Kinetic curve of formate dehydrogenase.Note: a) : Kinetics curve of formate dehydrogenase (CL8-FDH + Scaf) binding protein scaffold; b) : kinetics curve of formate dehydrogenase (CL8-FDH). Note: a): Kinetic curve of formate dehydrogenase binding protein scaffold; b): Kinetic curve of formate dehydrogenase.
(4) Effect of artificial protein scaffold system on HydABCC activity
[Fe-Fe] hydrogenase (HydABCC) catalyzes the reaction to produce hydrogen and NAD+ with NADH as substrate. In order to determine the effect of protein scaffold on the activity of the enzyme, the purified hydabCC was purified and the protein concentration was determined by Bradford method. The enzymatic kinetics curves of HydABCC and HydABCC+Scaf were plotted.
Figure. 7. Kinetics curve of [Fe-Fe] hydrogenase. Note: a) : Kinetics curves of [Fe-Fe] hydrogenase (HydABCC + Scaf) binding protein scaffold; b) : [Fe-Fe] hydrogenase (HydABCC) kinetic curve.
As shown in FIG. 7, the final concentration of NADH was 0.1mM. We determined the dynamic curve of substrate NADH with time under the catalysis of HydABCC and HydABCC +Scaf respectively. The addition of scaffold protein improved the catalytic efficiency and substrate specificity of HydABCC, and ruled out the possibility that scaffold protein had a negative effect on hydrogenase.
(5) The effect of artificial protein scaffold system on the regeneration system of coenzyme NADH
In the above individual enzyme activity measurement experiment, we found that scaffold protein can promote the activity of four enzymes, but whether the addition of scaffold protein will affect the overall activity of Coenzyme NADH regeneration system needs to be further explored. Therefore, methanol and NAD+ were used as substrates in this study. The NADH production of the single coenzyme NADH regeneration system (CL2-ADH, FALDH-CL7 and CL8-FDH) and the coenzyme NADH regeneration system combined with the protein scaffold (CL2-ADH, FALDH-CL7, CL8-FDH and Scaf-CIQ) was measured, respectively. The effect of scaffold protein on the overall enzyme activity of the coenzyme NADH regenerating system was analyzed. In the above individual enzyme activity measurement experiment, we found that scaffold protein can promote the activity of four enzymes, but whether the addition of scaffold protein will affect the overall activity of Coenzyme NADH regeneration system needs to be further explored. Therefore, methanol and NAD+ were used as substrates in this study. The NADH production of the single coenzyme NADH regeneration system (CL2-ADH, FALDH-CL7 and CL8-FDH) and the coenzyme NADH regeneration system combined with the protein scaffold (CL2-ADH, FALDH-CL7, CL8-FDH and Scaf-CIQ) was measured, respectively. The effect of scaffold protein on the overall enzyme activity of the coenzyme NADH regenerating system was analyzed.
Figure 8. Effect of scaffold protein on enzyme activity of Coenzyme NADH regenerating system. Note: Total: the amount of NADH produced by the Coenzyme NADH regeneration system combined with scaffold protein; Scaf- : NADH production from the Coenzyme NADH regeneration system alone.
(6) The influence of artificial protein scaffold system on hydrogen production system
Finally, the reaction efficiency of systems with and without scaffolds was compared by using Acar blue. It was found that the Aqua blue in the system with protein scaffold was lightened immediately after addition, while it took some time for the system without Aqua blue, as shown in Figure 9. This proves that the protein scaffold can promote the hydrogen production efficiency of the whole system.
Figure 9: Methylene blue test results
References
[1] Oroz-Guinea I, Garcia-Junceda E. Enzyme catalysed tandem reactions [J]. Current Opinion in Chemical Biology, 2013, 17(2): 236-49.
[2] Vazquez-Gonzalez M, Wang C, Willner I. Biocatalytic cascades operating on macromolecular scaffolds and in confined environments [J]. Nature Catalysis, 2020, 3(3): 256-73.
[3] Huang X, Holden H M, Raushel F M. Channeling of Substrates and Intermediates in Enzyme-Catalyzed Reactions [J]. Annual Review of Biochemistry, 2001, 70(1): 149-80.
[4] Huang, Xinyi, Holden, et al. CHANNELING OF SUBSTRATES AND INTERMEDIATES IN ENZYME-CATALYZED REACTIONS [J]. Annual Review of Biochemistry, 2001.
[5] Au - Meredith S, Au - Xu S, Au - Meredith M T, et al. Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization [J]. JoVE, 2012, (65): e3949.
[6] Schrittwieser J H, Velikogne S, Hall M, et al. Artificial Biocatalytic Linear Cascades for Preparation of Organic Molecules [J]. Chemical Reviews, 2017, 118(1).
[7] Saha A, Mondal G, Biswas A, et al. In vitro reconstitution of a cell-like environment using liposomes for amyloid beta peptide aggregation and its propagation [J]. Chemical Communications, 2013, 49(55): 6119-21.
[8] Xie J, Shen Q, Huang K, et al. Oriented Assembly of Cell-Mimicking Nanoparticles via a Molecular Affinity Strategy for Targeted Drug Delivery [J]. Acs Nano, 2019.
[9] Kozlovskaya V, Baggett J, Godin B, et al. Hydrogen-bonded Multilayers of Silk Fibroin: From Coatings to Cell-mimicking Shaped Microcontainers [J]. Acs Macro Letters, 2012, 2012(3): 384.
[10] Pinheiro A V, Han D, Shih W M, et al. Challenges and opportunities for structural DNA nanotechnology [J]. Nature Nanotechnology, 2011, 6(12): 763-72.
[11] Laurenti E, Barde I, Verp S, et al. Inducible Gene and shRNA Expression in Resident Hematopoietic Stem Cells In Vivo [J]. Stem Cells, 2010, 28(8): The 1390-8.
[12] Spivey H O, Ovadi J. Substrate channeling [J]. Methods, 1999, 19(2): 306-21.
[13] Liu F, Banta S, Chen W. Functional assembly of a multi-enzyme methanol oxidation cascade on a surface-displayed trifunctional scaffold for enhanced NADH production [J]. Chemical Communications, 2013, 49(36): 3766-8.
[14] Dueber J E, Wu G C, Malmirchegini G R, et al. Synthetic protein scaffolds provide modular control over metabolic flux [J]. Nature Biotechnology, 2009, 27(8): 753-9.
[15] Witkin E M. Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli [J]. Bacteriological reviews, 1977, 40(4): 869-907.
[16] Ko T P, Liao C C, Ku W Y, et al. The crystal structure of the DNase domain of colicin E7 in complex with its inhibitor Im7 protein [J]. Structure, 1999, 7(1): 91-102.
[17] Hsieh, S.-Y. A novel role of ImmE7 in the autoregulatory expression of the ColE7 operon and identification of possible RNase active sites in the crystal structure of dimeric ImmE7 [J]. Embo Journal, 2014, 16(6): 1444-54.
[18]Cole S T, Saint-Joanis B, Pugsley A P. Molecular characterisation of the colicin E2 operon and identification of its products [J]. 1985, 198(3): 465-72.
[19] To Ba M, Masaki H, Ohta T. Colicin E8, a DNase which indicates an evolutionary relationship between colicins E2 and E3 [J]. Journal of Bacteriology, 1988, 170(7): 3237-42.
[20] Tracy E, Richard J. Complete nucleotide sequence of the colicin E9 ( cei ) gene [J]. Nucl Acids Res, 1989.
[21] James E R, Moore J, Kleanthous C, et al. Protein-protein interactions in colicin E9 DNase-immunity protein complexes. 1. Diffusion-controlled association and femtomolar binding for the cognate complex [J]. Biochemistry, 1995, 34(42): 13743.
[22] Yang J, Wang F, Yang S, et al. Development of a Hyperthermostable Artificial Scaffold Based on Ultrahigh-Affinity Protein Pairs and Its Application in Cellulose Degradation [J]. ACS Sustainable Chemistry & Engineering, 2022, 10(6): 2072-83.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |